Hierarchical regulation of Burkholderia glumae type III secretion system by GluR response regulator and Lon protease

Abstract Expression of type III secretion system (T3SS) genes, which are important for the virulence of phytopathogenic bacteria, is induced in the plant apoplastic environment or artificially amended growth conditions. Wild‐type Burkholderia glumae BGR1, which causes rice panicle blight, induced a hypersensitive response (HR) in tobacco plants, whereas the T3SS genes were not significantly expressed in the commonly used hrp induction medium. T3SS gene expression in B. glumae was dependent on HrpB, a well known T3SS gene transcriptional regulator. Here, we report a stepwise mechanism of T3SS gene regulation by the GluR response regulator and Lon protease in addition to HrpB‐mediated control of T3SS genes in B. glumae. The gluR mutant showed no HR in tobacco plants and exhibited attenuated virulence in rice plants. GluR directly activated hrpB expression, indicating that hrpB belongs to the GluR regulon. The lon mutation allowed high expression of the T3SS genes in nutrient‐rich media. Lon directly activated gluR expression but repressed hrpB expression, indicating that Lon acts as a regulator rather than a protease. However, the lon mutant failed to induce an HR and virulence, suggesting that Lon not only acts as a negative regulator, but also has an essential, yet to be determined role for T3SS. Our results demonstrate the involvement of the two‐component system response regulator GluR and Lon in T3SS gene regulation, providing new insight into the complex interplay mechanisms of regulators involved in T3SS gene expression in bacteria–plant interactions.


| INTRODUC TI ON
The type III secretion system (T3SS) is a key virulence trait of many pathogenic bacteria and is highly conserved (Deng et al., 2017). The T3SS, which is encoded by a group of hypersensitive response and pathogenicity (hrp) genes, serves as a conduit for translocating a variety of effector proteins into plant cells, resulting in the induction of either a hypersensitive response (HR) in resistant hosts or in nonhost plants or pathogenicity in susceptible hosts (Alfano & Collmer, 1997;Balint-Kurti, 2019;Stam et al., 2014). Expression of T3SS genes is repressed under nutrient-rich conditions and is only induced under artificially amended conditions or in planta (Xie et al., 2019). Therefore, the T3SS is a sophisticated system with a very complex regulatory network. Assembly of the core components of T3SSs, needle length, substrate recruitment and secretion, and delivery of effector proteins are coordinately regulated in different bacterial pathogens (Deng et al., 2017). The expression of T3SS genes is transcriptionally regulated by diverse transcriptional regulators and strictly controlled by various external and host factors, such as temperature, pH, oxygen availability, and host-derived molecules (Hutcheson et al., 2001;Ortiz-Martin, Thwaites, Macho, et al., 2010;Xie et al., 2019). Two-component systems (TCSs) are involved in sensing T3SSinducing conditions and regulating hrp genes in the early stage of invasion (Xie et al., 2019). In Pseudomonas syringae, three TCSs, RhpRS, CvsRS, and GacAS, directly regulate the hrpRS-hrpL-T3SS cascade depending on the external nutrient conditions such as acetate or Ca 2+ concentrations (Xie et al., 2019). Conversely, RhpR phosphorylated by RhpS or acetyl phosphate activates gene expression of Lon protease to result in degradation of HrpR, a transcriptional activator of hrpL in P. syringae (Zhou et al., 2016).
Lon protease has been reported to play a negative role in T3SS gene regulation via degradation of the T3SS transcriptional regulator in Xanthomonas citri as well as P. syringae. In X. citri, Lon regulates the T3SS through proteolysis of HrpG depending on host-induced phosphorylation (Zhou et al., 2018). Phosphorylated Lon becomes inactive as a protease, resulting in the induction of the T3SS by HrpG-mediated regulation in X. citri (Zhou et al., 2018). In addition to the negative role of Lon in T3SS gene expression, Lon together with ClpXP induces the T3SS by cleaving YmoA, a repressor protein of the T3SS gene in Yersinia pestis (Jackson et al., 2004). Lon is also involved in the modulation of effector protein secretion as well as the assembly of the T3SS in P. syringae. The half-lives of several effectors such as AvrPto, HopPtoM, and HopPsyA are substantially higher in the lon mutant, suggesting rate-limiting effector secretion via Lonassociated degradation in P. syringae (Losada & Hutcheson, 2005).
We studied the pathogenic aspects of Burkholderia glumae, the causal agent of rice panicle blight, including toxoflavin and oxalate biosynthesis, quorum sensing (QS), QS-dependent motility and flagellar morphogenesis, and pellicle formation (Goo et al., 2012;Jang et al., 2014;Kim et al., 2004Kim et al., , 2007Kwak et al., 2020). This bacterium also relies on the T3SS for the successful infection of rice plants (Kang et al., 2008). However, the T3SS genes are not expressed in hrp-inducing conditions while being activated by HrpB, a major transcriptional activator of hrp gene expression (Kang et al., 2008). In this study, we found that hrp genes are highly expressed in the lon mutant and that the TCS response regulator GluR plays roles in hrp gene expression. These results allowed us to elucidate the regulatory networks mediated by HrpB, Lon, and GluR for hrp gene expression in B. glumae. We found that Lon protease acts as a regulator to activate gluR expression but repress hrpB expression, and GluR subsequently activates the expression of hrpB. These findings highlight another case of Lon acting as a transcriptional regulator rather than an ATPdependent protease. Our study demonstrates that the interplay of GluR and Lon along with the known regulator HrpB in controlling the expression of T3SS genes is critical for virulence in B. glumae.

| GluR was required for HR induction and full virulence of B. glumae
To assess whether this GluR-GluS TCS is involved in the virulence of B. glumae, the gluR mutant, the gluS mutant, and wildtype BGR1 were infiltrated into tobacco leaves and injected into the stems of rice plants. The gluS mutant and wild-type BGR1 induced an HR, while the gluR mutant failed to do so (Figure 1a).
The gluR mutant showed significantly reduced disease symptoms with an index of 0.34 ± 0.15 compared to wild-type BGR1 with a disease index of 1.0 ± 0.52 (Figure 1b). The complementation strain of the gluR mutant restored the HR and virulence as observed in wild-type BGR1 (Figure 1a,b). The disease index of the gluS mutant (0.73 ± 0.04) was comparable to that of wildtype BGR1 (Figure 1b), consistent with our previous findings that polycistronic gluR and gluS are not a functional pair (Marunga et al., 2021a). The viable cell numbers of wild-type BGR1, the gluR mutant, and the gluS mutant were similar for the 12 days after inoculation, indicating that mutations in gluR or gluS did not affect the colonization ability of B. glumae (Figure 1c). Both the gluR and the gluS mutants produced toxoflavin and QS signals at the same levels as wild-type BGR1 ( Figure S1).

| Mutation of gluR halted T3SS gene induction
Given that the gluR mutant showed no HR induction, we wondered whether GluR is involved in the regulation of T3SS genes in B. glumae. Because the hrp genes of B. glumae are not expressed in Luria-Bertani (LB) or hrp-inducing medium (Kang et al., 2008), we used LB supplemented with crude extracts of tobacco leaves to confirm the control of hrpB and hrpG expression by GluR. In LB medium amended with crude extracts of tobacco leaves, the hrpB and hrpG genes were expressed in wild-type BGR1 whereas the gluR mutant (BGLUR133) showed no detectable expression  Figure 2c). The katE promoter served as nonspecific competitor control DNA. In the upstream region of hrpB, we found a conserved inverted repeat sequence, comparable to those previously proposed as potential GluR-binding sites (Marunga et al., 2021a(Marunga et al., , 2021b Figure S2).

| A mutation in lon triggered T3SS gene expression but the lon mutant failed to induce an HR and disease symptoms
As we have previously reported that the T3SS genes of B. glumae are not expressed in artificially amended induction medium such as hrp induction medium (Kang et al., 2008), we expected that T3SS genes of B. glumae might be controlled in a different manner as compared to previously known mechanisms. During our study of the functional roles of Lon protease of B. glumae, we found that T3SS genes were highly expressed in the lon mutant in LB as assessed by RNA sequencing analysis (Table 1). We confirmed that the hrcC, F I G U R E 1 The response regulator GluR positively regulates the virulence of Burkholderia glumae BGR1. (a) Nonhost tobacco was inoculated with wild-type BGR1, the gluR mutant (gluR::Tn3-gusA133, BGLUR133), the gluS mutant (gluS::Tn3-gusA35, BGLUS35), and the gluR mutant complementation strain (gluR::Tn3-gusA133/gluR, BGLUR133C) and photographed 1 day after inoculation. SDW denotes sterile distilled water. (b) The stems of host rice plants were inoculated with the indicated B. glumae strains. Disease symptoms were photographed 7 days after inoculation; the numbers below the disease symptoms are the disease index scores relative to the wildtype values. Data represent the mean ± standard error of triplicate experiments. Superscripts (a and b) before the mean value indicate statistical significances (p < 0.05), F[8,11] = 22.047 (p < 0.01), based on one-way analysis of variance followed by Tukey's correlation for multiple comparisons. (c) Colonization was evaluated every 3 days after inoculation for 12 days by counting cfu of recovered cells from the infected rice stems. Data represent the mean ± standard error of triplicate experiments.

| Phenotypes of the gluR/lon double mutant
To determine a regulation hierarchy of T3SS genes mediated by GluR and Lon, we generated the gluR/lon double mutant and then evalu- Complementation of the lon gene in the gluR/lon double mutant did not affect the expression of T3SS genes ( Figure 4b).

| Regulation hierarchy of T3SS genes by GluR and Lon
Because Lon negatively regulated the expression of hrcC, hrpB, and

| DISCUSS ION
Unlike other plant-pathogenic bacteria possessing T3SSs such as P.
syringae, T3SS expression in B. glumae is not induced in hrp-inducing minimal medium (Kang et al., 2008). We did not understand why the hrp genes were not expressed in the induction medium, but studies of GluR and Lon protease in B. glumae revealed important clues. Our present study shows the first case where a TCS not clustered with T3SS genes is involved in the regulation of the hrp genes in B. glumae. The reduced virulence of the gluR mutant was not due to a lack of toxoflavin or QS signal production or a defect in colonization in rice ( Figure S1). We believe that the reduced virulence of the gluR mutant is a result of previously unidentified roles of GluR besides its involvement in normal cell division and β-lactam resistance in B. glumae (Marunga et al., 2021a(Marunga et al., , 2021b  However, expression levels of hrp genes in the wild type were not as high as we expected. This problem was solved by examining the expression level of the hrp genes in the lon mutant. Lon protease is a member of the ATPase associated with various cellular activities (AAA+) protease family and is highly conserved in prokaryotes and eukaryotes (Sauer & Baker, 2011). Lon contributes to diverse biological processes, including the heat shock response, drug resistance, DNA replication and repair, motility, and virulence factor production (Lan et al., 2007;Tsilibaris et al., 2006). Lon, which functions as a protease in P. syringae and X. citri, is also a regulator of the T3SS (Zhou et al., 2016;Zhou et al., 2018). With regard to the regulation of TCSs, there have been reports that the response regulator protein is degraded by proteases (Ogura & Tsukahara, 2010 glumae (Goo et al., 2017). Lon has also been shown to up-regulate and down-regulate various genes in P. syringae (Hua et al., 2020).
One puzzling question about the phenotype of the lon mutant was how the mutant lost the ability to induce an HR under high expression of hrp genes in the mutant. We propose two possible answers. One is that Lon might play another essential role in the proper functioning of each component in T3SS or T3SS-dependent proteins necessary for inducing the HR, as Lon has been reported to have a chaperone-like function (Shin et al., 2021). The other possibility is that the loss of the HR may be due to the growth defect of the lon mutant in vitro (Goo & Hwang, 2021).

| DNA manipulation and mutagenesis
Basic DNA manipulations were done following standard protocols (Sambrook et al., 1989). DNA sequencing was performed by Macrogen, Inc. (Seoul, Korea). The genetic information and gene IDs used in this study were obtained from the B. glumae BGR1 genome database (GenBank accession numbers CP001503-CP001508; kropb ase.snu.as.kr/cgi_bg.cg).

| Toxoflavin assay
Toxoflavin was extracted from overnight cultures using chloroform as previously described (Yoneda et al., 1971). Chloroform extracts were dissolved in dimethyl sulphoxide and applied to a silica gel 60 thin layer chromatography plate (Merck). Chromatograms were developed with chloroform/methanol (95:5, vol/vol). The spots were visualized under UV light at 365 nm.

| Autoinducer assay
The QS signal production assay was performed as previously described (Kim et al., 2004), with a few modifications. The acyl-homoserine lactones were extracted from overnight bacterial cultures by mixing the cell-free supernatant and ethyl acetate (1:1). The ethyl acetate extracts were evaporated using a rotary evaporator below 40°C, and the residues were reconstituted in 10 μl of dimethyl sulphoxide. A 5μl sample was then dropped on LB agar medium containing a Chromobacterium violaceum biosensor, and the plates were incubated at 28°C overnight.

| Preparation of plant extracts
Tobacco leaves that were 4 weeks old were used to make crude plant extract. The leaves were washed under running water and dried at 37°C, and dry weight was determined before being crushed into powder. The powder was soaked in 95% methanol F I G U R E 6 Schematic representation of the hierarchy of regulatory mechanisms of the type III secretion system (T3SS) by Lon and GluR in Burkholderia glumae BGR1. Regulatory pathways are proposed based on our observations. The present study showed regulation mediated by the two-component system response regulator GluR and Lon protease in the T3SS.

| RNA extraction and RT-qPCR
Total RNA was isolated from B. glumae strains and cDNA was synthesized as previously described (Marunga et al., 2021a) using Recombinant RNasin and M-MLV reverse transcriptase following the manufacturer's instructions (Promega). Using primers listed in  (Li & Durbin, 2009). The mRNA reads were normalized to reads per kilobase per million mapped reads (Mortazavi et al., 2008). The NCBI SRA accession number for the RNA sequencing data series of BGR1, BLONN, and BLONC is PRJNA727974.

| EMSA
GluR-His and His-Lon-His were purified as described previously (Goo & Hwang, 2021;Marunga et al., 2021a). Using primer sets hrpBp-F/R and glurp-F/R, listed in Table S2, putative promoter regions of the respective genes were amplified and labelled with biotin using Lightshift Chemiluminescent Electrophoretic Mobility Shift Assay Kits, as described by the manufacturer (Pierce). The EMSAs were performed as previously described (Kim et al., 2007). The putative promoter region of katE1 (329 bp) was used as nonspecific competitor DNA, and band detection was done following previously described methods (Marunga et al., 2021a).

| Protein in vitro degradation assay
The degradation assay was performed as previously described (Zhou et al., 2018)

| Statistical analyses
All experiments were conducted in triplicate with the respective controls. One-way analysis of variance (ANOVA) was used followed

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data sets generated for this study are included in this article or supporting information; further inquiries can be directed to the corresponding author.